In utero hematopoietic stem cell transplantation (IUSCT) is a promising therapeutic approach for many genetic disorders. However, after almost two decades, the only applications for which IUSCT has proven successful are diseases that confer a selective advantage on donor cells. This suggests that the basic concepts used as rationale for IUSCT need to be revisited so that strategies to achieve therapeutic levels of engraftment can be developed. In the last funding period, we attempted to improve donor cell engraftment by co-transplanting marrow stromal cells with hematopoietic stem cells (HSC), to establish a donor friendly environment in the fetal host. The results obtained were encouraging, but the observed levels of donor cell engraftment would not be therapeutic for many clinical applications. Furthermore, since few stromal cells were detected in the fetal bone marrow (BM) post-transplantation, the enhancement of donor cell engraftment was likely due to an immunomodulatory effect of the stromal cells rather than the direct contribution of stromal cells to the fetal microenvironment. For this reason, we developed the necessary techniques/reagents to perform transplants with matched allogeneic umbilical cord blood (UCB) HSC, and significantly increased the levels of donor cell engraftment in both BM and peripheral blood, demonstrating that the fetal immune system plays a key role in fetal transplantation and that donor-recipient matching may be important in achieving cure after IUSCT. Our data also suggests that optimization of the HSC transplantation timing is needed to ensure that receptive microenvironmental niches are present in the BM to support the long-term engraftment and differentiation of the transplanted cells. The central hypothesis of this request for competing renewal application is that targeting the fetal immune-response and ensuring the presence of a supportive microenvironment will enable achievement of therapeutic levels of HSC engraftment following IUSCT and cure a clinically relevant sheep model of a lysosomal storage disease. To test this hypothesis, we will: 1) determine when during gestation the hematopoietic niche and the resident CD34+ HSC become fully functional;2) use cell grafts composed of matched allogeneic UCB HSC alone or in conjunction with same donor immunomodulatory cells to establish donor-specific immune tolerance to defeat the adaptive host immune system;and 3)induce tolerance to donor cells early in gestation to enable a later same donor HSC transplant, thus targeting a mature BM environment supportive of long-term engrafting donor HSC capable of myelopoietic differentiation. We will next apply the same IUSCT strategy to a sheep model of Batten's disease as proof of principle that IUSCT can be used to cure this fatal, inherited disorder of the nervous system prior to birth. PUBLIC HEALTH RELEVANCE In utero hematopoietic stem cell transplantation (IUSCT) is a promising therapeutic approach for many inherited diseases. However, after almost two decades, the only applications for which IUSCT has proven successful are diseases that confer a selective advantage on donor cells. The overall goal of this proposal is to employ specific strategies targeting both the immune-response and the need for supportive microenvironmental space in the fetus so that therapeutic levels of donor hematopoietic stem cell engraftment following pre-natal transplantation can be achieved. We will then prove the validity of our approach in a clinically relevant sheep model of a lysosomal storage disease.